Cloning and analysis of a Y-tocopherol methyltransf erase gene from Brassica oleracea and the function of its recombinant protein

The full-length cDNA (BoTMT) of γ-TMT is obtained from Brassica oleracea by 3'- and 5'-RACE methods. The 1265 bp cDNA contains an open reading frame of 1044 bp, which encodes a protein of 347 amino acids with a predicted chloroplast transit peptide and two S-adenosylmethionine (SAM)-binding domains. Sequence analysis shows that the deduced protein shares 41. 8% -86. 5% similarity to known γ-TMTs. Semi-quantitative RT-PCR reveals that BoTMT is expressed preferentially in flowers and leaves of B. oleracea . The recombinant γ-TMT protein is obtained by cloning its encoding region into the prokaryotic expression vector pET30a. The protein expressed in E. coli accounts for 22 % of total bacterial protein. The enzyme activity assay indicates that the recombinant protein has relatively high activity to convert γ-tocopherol to α-tocopherol in vitro.

Cloning and analysis of a Y-tocopherol methyltransf erase gene from Brassica oleracea and the function of its recombinant protein